The goal of this proposal is to understand why the majority of melanoma patients fail to respond to targeted inhibition of the Raf-MEK-ERK pathway even though inhibition of this pathway prevents the growth of melanoma cell lines in cell culture and in animal models. Two hypotheses explaining the resistance of melanomas to targeted MEK inhibition are 1) that drug fails to accumulate at the site of the tumor or 2) that signals from the tumor microenvironment prevent melanoma cells from relying on MEK signaling for survival. To investigate these possible mechanisms of resistance, we will use an innovative mass spectrometry method to profile MEK-dependent phosphorylation events in a number of metastatic melanoma cell lines using the MEK1/2 inhibitor AZD6244. By comparing MEK-dependent phosphorylation events in these cell lines, we will identify phosphosites that are invariably regulated by the Raf-MEK-ERK pathway in melanoma cell lines, establishing a melanoma "phosphosignature". We will also generate quantitative dose response curves for each of these sites. We will then make use of a novel xenograft model of melanoma, in which patient samples show resistance to MEK inhibition in vivo, and compare the phosphosignature of AZD6244-treated tumors to the phosphosignature established in cell lines. By comparing these signatures, it will be possible to determine the effective dose of drug that accumulates at resistant melanomas and determine if the tumor microenvironment affects Raf-MEK-ERK signaling in these tumors. Thus, these studies will use pioneering methods in molecular biology and mass spectrometry to improve our understanding of cell signaling and cancer treatment. PUBLIC HEALTH RELEVANCE: The majority of melanomas harbor activating mutations in the Raf-MEK-ERK signaling pathway and inhibition of this pathway causes cell death in melanoma cell lines. However, for reasons that remain poorly understood, most advanced stage melanoma patients fail to respond to drugs that inhibit this pathway. The proposed study will investigate the mechanisms of resistance to the MEK inhibitor AZD6244 in metastatic melanoma.